WO2009152452A1 - Procédé de fermentation pour la production de protéines neurotoxines botuliques solubles - Google Patents

Procédé de fermentation pour la production de protéines neurotoxines botuliques solubles Download PDF

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WO2009152452A1
WO2009152452A1 PCT/US2009/047240 US2009047240W WO2009152452A1 WO 2009152452 A1 WO2009152452 A1 WO 2009152452A1 US 2009047240 W US2009047240 W US 2009047240W WO 2009152452 A1 WO2009152452 A1 WO 2009152452A1
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culture
induction
temperature
neurotoxin
soluble
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PCT/US2009/047240
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Huei-Hsiung Yang
Ian Mcentee
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Huei-Hsiung Yang
Ian Mcentee
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Publication of WO2009152452A1 publication Critical patent/WO2009152452A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)

Definitions

  • the present invention relates to methods for producing soluble recombinant botulinum neurotoxin proteins using bacterial fermentation.
  • Bacterial toxins and toxin fragments are importation as immunodiagnostic reagents, therapeutic agents, and vaccine components.
  • One group of bacterial toxins is the clostridial neurotoxins, such as those from Clostridium botulinum and Clostridium butyricum.
  • Botulinum neurotoxin (BoNT) is one of the most potent toxins known to man. Its ingestion or inhalation inhibits neurotransmitter release from synaptic vesicles, resulting in neuroparalysis and death.
  • BoNT Botulinum neurotoxin
  • the use of Clostridium botulinum neurotoxins as vaccine components is disclosed in U.S. Patent No. 5,919,665 to J. A. Williams, which is incorporated by reference into this application.
  • the botulinum neurotoxins are a family of seven antigenically different protein toxins (serotypes A, B, C, D, E, F, and G). Antisera raised against purified neurotoxins show no cross-protection between the neurotoxin serotypes.
  • neurotoxins are extremely potent neuroparalytic agents that act primarily at the peripheral nervous system where they inhibit the release of acetylcholine at the neuromuscular junction (Niemann (1991) In Sourcebook of Bacterial Protein Toxins (Alouf, J. E. & Freer, J. H. eds.), pp. 303-348, Academic Press, London). This activity is mediated via highly specific zinc-dependent endopeptidase activity directed at small proteins involved in the fusion and release of synaptic vesicles.
  • the botulinum neurotoxins are structurally similar, with 30-40% sequence homology.
  • Each neurotoxin type shares a common architecture in which a catalytic L-chain (LC, ⁇ 50 kDa) is disulfide linked to a receptor binding and translocating H-chain (HC, ⁇ 100 kDa).
  • LC catalytic L-chain
  • Hc fragment The C- terminal 50 kDa fragment (Hc fragment) is responsible for receptor-binding at the presynaptic nerve surface (Halpern & Loftus (1993) J. Biol. Chem. 268, 11188-11192); (Shone et al. (1985) Eur. J. Biochem., 151 , 75-82).
  • the N- terminal 50 kDa portion of the heavy chain (HN fragment) is involved in translocation of the enzymatically active light chain to within the nerve terminal (Shone er a/. (1987) Eur. J. Biochem., 167, 175-180).
  • Removal of the Hc domain from the BoNT leaves a fragment (LH N , ⁇ 100 kDa) consisting of the light chain and translocation domain which, although virtually non-toxic, is stable and relatively soluble. Residual toxicity can be eliminated, for instance, by introducing single or double mutations into the enzymatic domain, which yield a non-toxic LHN fragment that can be used in vaccine development.
  • rLH N fragments When they are expressed as recombinant (r) proteins, rLH N fragments have a tendency to form aggregates that may have a molecular mass ranging up to several million kilodaltons (kD). Although aggregated rLH N can be recovered from insoluble lysate material by detergent extraction/reductant treatment and further purified ( ⁇ 90%) by anion exchange (Q Sepharose) and gel filtration (Superdex 200) chromatography, the recovered aggregate has undesirable properties.
  • Q Sepharose anion exchange
  • Superdex 200 gel filtration
  • ⁇ LHN/E purified, aggregated ⁇ LHN from serotype E
  • BoNT/E native serotype E neurotoxin
  • animal efficacy data indicate that immunization with aggregated LhWE does not protect animals against BoNT/E toxin challenge.
  • the relative amount of ⁇ LHN produced in soluble form versus insoluble form during the fermentation process is a contributing factor to the development of aggregates. Applicants have observed that when the ratio of soluble:insoluble LH N protein is low (i.e., less of the LH N protein is in a soluble form) during fermentation, then the greater the tendency of the LH N to form aggregates, both during the fermentation process itself and during downstream isolation procedures. [010]
  • One method for determining if a protein is expressed in a soluble form during fermentation is to lyse the cultured host cells containing the expressed protein, centrifuge the cell lysate at 25,000xg for 15 minutes to pellet cell debris and aggregated protein material, then analyze the supernatant for presence of the protein.
  • Soluble proteins are found in the supernatant, whereas the pellet contains the insoluble proteins and protein aggregates.
  • SDS-PAGE followed by a detection method such as Coomassie staining or western blotting, can be used to estimate the amount of protein in each fraction.
  • a detection method such as Coomassie staining or western blotting
  • the disclosure provides methods for increasing the proportion or total amount of soluble Clostridium neurotoxin and neurotoxin fragments (for instance, recombinant LH N ) during the fermentation process, thereby reducing or preventing aggregate formation during the production of the neurotoxin.
  • the fragment is a rl_H N fragment (a non-toxic derivative of Clostridium botulinum or Clostridium butyricum neurotoxin A, B, C, D, E, F, or G).
  • the LH N fragment is a recombinant fragment chosen from ⁇ LHN/A, ⁇ LHN/B, or ⁇ LHN/E.
  • the Clostridium neurotoxin fragment comprises LH N and a portion of the Hc domain as provided in WO 2007/044382, which is herein incorporated by reference in its entirety.
  • the Clostridium neurotoxin fragment is further modified or fused to one or more additional peptides as disclosed in WO 2004/024909, which is herein incorporated by reference in its entirety.
  • the invention includes a method for increasing soluble Clostridium neurotoxin or neurotoxin fragment (e.g., rl_H N ) production during fermentation by a novel method of auto-induction.
  • the invention comprises growing a host cell that has been transformed with a nucleic acid encoding a neurotoxin or neurotoxin fragment (e.g., recombinant LH N fragment) in a growth-supporting media until the culture reaches a designated OD 6 oo or saturation density, and reducing the temperature of the culture below about 25° C.
  • the growth-supporting media is a media that supports auto-induction of synthesis of the recombinant LHN fragment. That is, in some embodiments the media is formulated such that it is not necessary to add an exogenous induction agent, such as isopropylthio- ⁇ -D- galactoside (IPTG), in order to induce expression of the recombinant LH N fragment.
  • an exogenous induction agent such as isopropylthio- ⁇ -D- galactoside (IPTG)
  • the Clostridium toxin or toxin fragment is grown in growth-supporting media at about 30° C to 40° C until the culture reaches a designated OD 6 oo- In one embodiment, the toxin or toxin fragment is grown in media at about 35° C to 40° C prior to induction. In one embodiment the designated OD 60O is about 15 to 20.
  • the temperature of the culture is reduced after the culture reaches the designated OD 6 oo or saturation density.
  • the temperature of the culture is reduced to about or less than 20° C for induction.
  • the invention includes a method for increasing soluble rLH N production during fermentation by reducing the temperature of the culture to about 10° C , 11 0 C , 12° C, 13° C, 14° C, 15° C, 16 0 C, 17° C, 18° C, or 19° C.
  • the temperature of the culture is reduced to about 15° C to about 20° C.
  • the method for increasing soluble Clostridium toxin or toxin fragment further comprises reducing the dissolved oxygen in the culture after the culture reaches the designated OD 6OO -
  • the invention includes methods of increasing soluble peptide production by reducing the dissolved oxygen in the culture by at least about 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, or 25 fold compared to the dissolved oxygen of the culture prior to induction (for instance, at the designated OD ⁇ oo or saturation density).
  • the invention includes reducing the dissolved oxygen of the culture by reducing the agitation of the culture.
  • the reduction of agitation of the culture can occur at or about the same time as the reduction in temperature.
  • the invention includes, for instance, a method for increasing soluble ⁇ LHN comprising reducing the dissolved oxygen in the culture to about 0 to 10% DO and reducing the temperature of the culture below about 25° C.
  • the invention includes a method for increasing soluble ⁇ LH N comprising reducing the dissolved oxygen in the culture to about 0 to 5% DO and reducing the temperature of the culture to about 15° C to 17° C.
  • the method for increasing soluble Clostridium toxin and toxin fragment production further comprises, after reducing the temperature of the culture, growing the host cells for an additional period of time under conditions that permit expression of the gene.
  • the cells are cultured for an additional about 10 to 25 hours, about 15 to 25 hours or about 15 to 20 hours.
  • the cells are cultured an additional about 16 to 20 hours at about 16° C.
  • Various embodiments of the method further comprise isolating the Clostridium toxin or toxin fragment (e.g., ⁇ LHN) expressed during the culture.
  • Expressed toxins or toxin fragments can be isolated by methods generally known in the art, for instance, SDS-PAGE.
  • the Clostridium toxin or toxin fragment (e.g., ⁇ LHN) resulting from the auto- induction methods of the present invention is purified.
  • the methods of the invention can be used to produce toxins and toxin fragments that exhibit reduced aggregation as compared to toxins and toxin fragments expressed in the same host cell under conditions that do not result in auto-induction.
  • the amount of aggregation can be determined by methods known in the art, for instance, SDS-PAGE.
  • Figures 1A and 1B show a Coomassie Blue stained gel and western blot analysis, respectively, of ⁇ LHN/A fragments in different cellular fractions before and after induction of protein expression.
  • Figure 2A and 2B also show a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H N /A fragments expressed in E. coli strain ER2566 following induction of protein expression at different temperatures.
  • Figure 3 is a graph summarizing the amounts of rl_H N /A found in the soluble versus insoluble fractions.
  • Figure 4A and 4B present a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H N /A fragments expressed in E. coli strain BL21 following induction of protein expression at different temperatures.
  • Figure 5 is a graph summarizing the amounts of ⁇ LHN/A found in the soluble versus insoluble fractions.
  • Figure 6A and 6B are fermentation profiles that plot various culture parameters on the Y-axis against time on the X-axis.
  • Figure 7A and 7B are Coomassie stained gel and western blot analysis, respectively, of the soluble fraction of cell lysates collected at approximately 20 to 21 hours post-induction in a 5 liter fermentor.
  • Figure 8A and 8B are fermentation profiles that plot various culture parameters on the Y-axis against time on the X-axis for two fermentations of E. coli ER2566 (pET26b-LH N E (OPT-) in a working volume 10-liter fermentor.
  • Figure 9A and 9B present a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H N /B fragments expressed by E. coli ER2566 (pET26b- rl_H N B-H(-)-L(-) following induction of protein expression at different temperatures.
  • Figure 10 is a fermentation profile plotting various culture parameters on the Y-axis against time on the X-axis for E. coli ER2566 (pET26b- rl_H N B-H(-)-L(-) in a 50-liter fermentor in which the induction protocol was applied.
  • BoNT botulinum neurotoxin. When a letter follows this designation, the letter indicates the serotype. For example, BoNT/A is botulinum neurotoxin type A.
  • LHN a fragment of a clostridial neurotoxin (including, but not limited to, botulinum or tetanus) of approximately 10OkDa which may be a single-chain or di-chain molecule comprising the light chain and the H N domain. The latter domain represents the N-terminal 5OkDa of the neurotoxin heavy chain and is closely associated with the light chain domain in the fragment.
  • LHN fragments including LH N /A and LH N /E, are described in United States Provisional Application Nos. 60/929,125, filed June 14, 2007, and 60/960,771 , filed October 12, 2007, the entire disclosure of each of which is relied upon and incorporated by reference. Additional examples include those described in U.S. Patent No. 6,461 ,617 to Shone et al.
  • LH N fragments may include those fragments possessing, as well as and those fragments lacking, the initial methionine.
  • LHN fragments include those that have been modified to render them endopeptidase negative. This may be accomplished by mutating the HELIH active site motif found in serotypes A, B, E, F, G, and tetanus neurotoxin to, for example, HQLIY.
  • the native motif HELNH may be mutated to, for example, HQLNY.
  • the active site motif HELTH may be mutated to, for example, HQLTY.
  • Endopeptidase negative displays no endopeptidase activity by conventional assays.
  • a Clostridium neurotoxin or neurotoxin fragment e.g., LHN
  • a Clostridium neurotoxin or neurotoxin fragment is made endopeptidase negative by modifying one or two or more amino acids by methods known in the art.
  • Amino acid modifications that confer an endopeptidase negative phenotype include, but are not limited to modifications at residues E224 and/or H227 for C. botulinum type A neurotoxin or fragments such as LH N /A, residues E231 and/or H234 for C.
  • botulinum type B neurotoxin or fragments such as LH N /B, and residues E213 and/or H216 for C. botulinum type E neurotoxin or fragments such as LHN/E.
  • the invention includes LH N /A comprising E224Q and/or H227Y modifications, LH N /B comprising E231Q and/or H234Y modifications and LHN/E comprising E213Q and/or H216Y modifications.
  • Auto-induction a fermentation technique in which over- expression of a target protein in a host is induced by reduction of dissolved oxygen (DO) and reduction of temperature. Auto-induction can be performed with small and large culture volumes.
  • the target protein is expressed from a nucleic acid on a plasmid in a bacterial cell.
  • cells are grown in a fermentor and induction occurs by (1) by reducing the agitation of cells within the fermentor (which results in a reduction of dissolved oxygen) and (2) reducing the temperature within the fermentor.
  • Auto-induction does not require the addition of a conventional inducer, such as IPTG, during the culture process.
  • a conventional inducer such as IPTG
  • auto-induction can be induced by contacting the cells with an inducer such as IPTG and reducing the temperature of the culture.
  • Soluble a "soluble" recombinant protein is one that exists in solution in the cytoplasm of the host cell. If the protein contains a signal sequence the soluble protein is exported to the periplasmic space in bacteria hosts and is secreted into the culture medium in eukaryotic cells capable of secretion or by bacterial host possessing the appropriate genes. In contrast, an insoluble protein is one that exists in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell.
  • a soluble protein is a protein that is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.
  • a soluble protein is distinct from a "solubilized" protein.
  • An insoluble recombinant protein found inside an inclusion body may be solubilized (i.e., rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold).
  • denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS).
  • proteins that are soluble i.e., dissolved
  • a solution devoid of significant amounts of ionic detergents e.g., SDS
  • denaturants e.g., urea, guanidine hydrochloride
  • proteins that exist as a suspension of insoluble protein molecules dispersed within the solution A soluble protein will not be removed from a solution containing the protein by centrifugation using conditions sufficient to remove bacteria present in a liquid medium (e.g., centrifugation at 5,00Og for 4-5 minutes).
  • a method of testing whether a protein is soluble or insoluble is described in U.S. Patent No. 5,919,665.
  • Dissolved Oxygen refers to the amount of oxygen dissolved in the culture and is typically expressed as percent DO.
  • aeration is set at about 0.5 or 1.0 WM, agitation at about 200 to 400 rpm, and DO calibrated to about 100%.
  • oxygen is utilized and the DO decreases.
  • the DO drops to about 30% at which point, agitation is increased to maintain a 30% DO until the culture reaches the designated cell density and/or growth phase (e.g., about 15-20 OD 6 oo).
  • agitation is decreased (e.g., to about 200 to 400 rpm) which, in turn, causes the DO to decrease to about 0-5%.
  • Auto-induction can be achieved by reducing the DO of a culture while also reducing the temperature of the culture.
  • auto- induction can be achieved in the present invention by reducing the DO of the culture by at least about 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold or 50 fold or more compared to the DO of the culture prior to auto-induction.
  • the invention provides methods of increasing soluble Clostridium toxin or toxin fragments (e.g., ⁇ LHN) production during the culture process.
  • the culture method results in the production of more soluble protein than insoluble protein.
  • the soluble rLH N protein is at least 60% of total rl_H N protein, although it may also be at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or even at least 98% of the total rl_H N protein produced in that culture.
  • rl_H N protein it is the total amount of rl_H N protein that increases. Increases may be measured relative to a culture process in which the induction temperature remains above 25 0 C or above 20 0 C. Alternatively, increases may be measured by comparison to a culture process that employs a growth-supporting media that does not comprise both lactose and a carbon source. It is also possible for the method to result in an increase in both the relative percentage of soluble protein and the total amount of soluble protein.
  • Clostridium neurotoxins or neurotoxin fragments including, but not limited to, BoNT/A, BoNT/B, BoNT/E, BoNT/F, BoNT/G, tetanus, LH N /A, LH N /B, LH N /E, LH N /F, LH N /G, LH N tetanus, Clostridium neurotoxins and fragments disclosed in WO 2007/044382 and WO 2004/024909, and fusion proteins comprising a Clostridium neurotoxin or toxin fragment (e.g., his-tagged LHN).
  • a suitable cell line is transformed with a DNA sequence encoding the neurotoxin or toxin fragment under the control of known regulatory sequences.
  • Genes encoding botulinum neurotoxins are available with codon bias appropriate for expression in a variety of host cells, and any of a wide variety of host cells are suitable for expression of a Clostridium neurotoxin or toxin fragment (e.g., rl_H N fragment).
  • bacterial cells are hosts cells for expression of the Clostridium toxin or toxin fragment (e.g., rLHN).
  • Clostridium toxin or toxin fragment e.g., rLHN
  • various strains of E. coli e.g., BL21(DE3), ER2566, HB101 , MC1061 are well-known as host cells in the field of biotechnology.
  • Various strains of B. subtilis, Pseudomonas, other bacilli, and the like may also be used.
  • DNA encoding the propeptide is generally not necessary.
  • the host cells used in the fermentation method of the present invention are generally a strain of Escherichia coli.
  • the E. coli strain expresses in its chromosome the coding sequence for T7 RNA polymerase under the control of an inducible lacUV5 promoter.
  • Non-limiting examples of E. coli strains that are suitable for use in auto-induction methods include ER2566 and BL21 (DE3).
  • suitable host cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO), the monkey kidney COS-1 cell line, or mammalian CV-1 cells.
  • CHO Chinese hamster ovary cells
  • CV-1 mammalian CV-1 cells.
  • Yeast cells such as Pichia pastoris may also be used.
  • the expression vector encoding the Clostridium neurotoxin or toxin fragment can vary widely in its components, but they will include those regulatory elements appropriate to the host cell selected.
  • the neurotoxin or neurotoxin fragment (e.g., LHN fragment) may be under the control of a promoter, for instance, a T7 promoter.
  • a promoter for instance, a T7 promoter.
  • various types of vectors can be used to express the Clostridium neurotoxin and fragments using the methods of the invention.
  • the expression vector includes, but is not limited to, pET26b (Novagen).
  • Nucleic acid sequences encoding various botulinum neurotoxins have been cloned and those nucleic acid sequences are known in the art.
  • BoNT/E from C. botulinum is provided in GenBank accession no. AB082519.
  • a nucleic acid sequence of a full length neurotoxin E from C. butyricum is provided in GenBank accession no. AB088207.
  • Methods of manipulating nucleic acids and of expressing the encoded proteins are known in the art, and include those described in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and Current Protocols in Molecular Biology (Eds.
  • the invention also encompasses LH N fragments in which the solubility has been improved by modifications to the amino acid sequence.
  • solubility-improving modifications are described in WO 2007/044382.
  • certain cysteine residues in the LHN fragment may be replaced with another amino acid that does not form a disulfide bond, such as serine.
  • solubility may be improved by extending the sequence of an LHN fragment by providing additional sequences from an adjoining segment, such as the Hc fragment.
  • the vector encodes an LHN fragment that is expressed as a fusion protein with, for example, a tag to increase the stability of the resulting fusion protein or to simplify purification.
  • tags are known in the art. Representative, non-limiting, examples include sequences that encode a series of histidine residues, the epitope tag FLAG, the Herpes simplex glycoprotein D, beta-galactosidase, maltose binding protein, streptavidin tag or glutathione S-transferase.
  • the bacterial cells are cultured on a growth-supporting media that supports auto-induction of synthesis of the recombinant LHN fragment from the expression vector.
  • the media is formulated such that it is not necessary to add an exogenous induction agent, such as isopropylthio- ⁇ -D-galactoside (IPTG), in order to induce expression of the recombinant LH N fragment. That is, when cultured in a medium containing lactose, there is little expression the LH N fragment at early stages of the culture, but protein expression is turned on automatically at a later (high-density or saturation) stage without any intervention.
  • IPTG isopropylthio- ⁇ -D-galactoside
  • a strain of E. coli that expresses the coding sequence for T7 RNA polymerase under the control of an inducible promoter, for instance, lacUV ⁇ promoter, and contains a vector containing the recombinant LH N fragment under the control of the T7 promoter is inoculated into culture medium.
  • the medium can vary in its constituents, but it should include lactose and at least one other carbon source. It should also be buffered such that, even at saturation, the pH of the culture remains near neutral. The cells are cultured without intervention and allowed to reach saturation.
  • Saturation density can be determined by measuring the optical density (OD) of the culture.
  • OD monitoring of the culture is performed by taking a culture sample from the fermentor, diluting the sample with Dl water or saline buffer, and measuring the diluted sample at 600 nm wavelength using a UV/Vis or Vis spectrophotometer.
  • Saturation densities often have an OD 6 oo range of around 10-20, but may reach as high as 30-50. However, lower saturation densities, such as around 5, may occur if the expressed protein is one that affects cell growth.
  • the OD of the culture is allowed to reach about 15-20 OD 6 oo before the induction phase is initiated.
  • the induction phase is initiated by the reduction of temperature of the culture and reduction of the dissolved oxygen in the culture.
  • agitation of the culture is reduced to reduce the dissolved oxygen content of the culture to about 0-5%DO.
  • the reduced temperature and percent dissolved oxygen of the culture are maintained.
  • the induction period typically lasts at least about 10 hours, about 12 hours, or about 15 hours. However, as can be appreciated by a skilled artisan, the induction period can vary considerably based on the desired amount of soluble Clostridium neurotoxin protein or protein fragment (e.g., LHN).
  • the incubation temperature of the fermentation culture during the induction phase affects the total amount of soluble Clostridium toxins or toxin fragments (e.g., rl_H N ) that is expressed, as well as the relative proportion of soluble to insoluble ⁇ LHN fragments. Accordingly, in some embodiments, the methods involve induction at a temperature that is lower than the initial culture temperature. In certain embodiments, induction is via auto-induction. In other embodiments, induction is by conventional methods, such as addition of IPTG.
  • the methods of the invention generally employ an induction temperature that is between about 15°C and about 35°C.
  • the minimum induction temperature is typically less than or about 15 0 C, about 16 0 C, about 17 0 C, about 18 0 C, about 19 0 C, about 2O 0 C, about 25 0 C, or about 3O 0 C.
  • the induction temperature may also be expressed as a range.
  • some embodiments involve an induction temperature of between about 15 to about 35 0 C, between about 15 to about 3O 0 C, between about 15 to about 25 0 C, between about 15 to about 2O 0 C, between about 15 to about 19 0 C, between about 15 to about 18 0 C, between about 15 to about 17 0 C, and between about 15 to about 16 0 C.
  • the induction temperature is a range of between about 16 to about 35 0 C, between about 16 to about 3O 0 C, between about 16 to about 25 0 C, between about 16 to about 2O 0 C, between about 16 to about 19 0 C, between about 16 to about 18 0 C, and between about 16 to about 17 0 C.
  • the induction temperature is a range of between about 17 to about 35 0 C, between about 17 to about 3O 0 C, between about 17 to about 25 0 C, between about 17 to about 2O 0 C, between about 17 to about 19 0 C, and between about 17 to about 18 0 C. In yet other embodiments, the induction temperature is a range of between about 18 to about 35 0 C, between about 18 to about 3O 0 C, between about 18 to about 25 0 C, between about 18 to about 2O 0 C, and between about 18 to about 19 0 C.
  • Still other embodiments involve an induction temperature that is in a range of between about 19 to about 35 0 C, between about 19 to about 3O 0 C, between about 19 to about 25 0 C, between about 19 to about 2O 0 C, between about 20 to about 3O 0 C, and between about 20 to about 25 0 C. In some embodiments, the induction temperature is about 16 0 C.
  • the methods of the present invention include reducing the amount of dissolved oxygen to stimulate auto-induction.
  • the amount of dissolved oxygen can be reduced at or about the time the temperature of the culture is reduced, to increase the ratio of soluble protein to insoluble protein expressed by the host cell.
  • the cell culture is grown at a level of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% dissolved oxygen prior to induction.
  • the level of dissolved oxygen decreases from about 100% to 30%.
  • the dissolved oxygen level gets to about 30%, the dissolved oxygen level is maintained by increasing agitation.
  • agitation is decreased so that the dissolved oxygen level decreases.
  • the dissolved oxygen level can be decreased to as little as about 0-5% DO.
  • the amount of dissolved oxygen at induction and continuing during the induction period can be about 0-3%, 0-5%, 0-10%, 5-10%, 5-15%, 0-15%, or less than 20% DO.
  • Neurotoxic proteins and fragments of those proteins are important immunodiagnostic reagents, therapeutic agents, and vaccine components. Functional neurotoxins are hazardous to work with, however, so investigators prefer to use recombinant proteins that have been genetically modified to reduce or eliminate their neurotoxicity. Unfortunately, it can be difficult to purify some of the recombinant, non-toxic, proteins because they often form aggregates, which have reduced solubility and are less effective reagents for use in immunodiagnostic, therapeutic, and vaccine applications.
  • the disclosure provides methods for reducing or preventing the formation of aggregates during the fermentation process.
  • the toxin fragments of the invention can be used as therapeutic agents and vaccine components. Any animal that is susceptible to the wild- type toxin can be vaccinated with the toxin fragment in an immunostimulatory composition. Accordingly, a vaccine composition comprising a clostridial neurotoxin rl_H N fragment can be used to protect rabbits, rodents, birds, horses, cattle, and humans, including infant humans, from botulism, or from one or more of the symptoms of botulism, such as diarrheal disease, paralysis (either mild or severe), or death.
  • the immunogenicity of the recombinant proteins can be tested by immunizing mice with, for example, 10 ⁇ g of LH N protein suspended in an adjuvant emulsion.
  • Control mice are immunized with saline emulsified in adjuvant for use as negative controls.
  • the mice are immunized i.p. four times at 2-week intervals.
  • One week after the last immunization the mice are bled and the serum is analyzed by immunoblot for the presence of specific antibody.
  • ELISA is used to determine the titer of the antisera.
  • each mouse is challenged i.p. with 2 lethal doses of the corresponding BoNT protein. Four days after challenge, the mice are scored for survivors.
  • Toxin fragments can also be used to prepare compositions comprising neutralizing antibodies that immunoreact with the wild-type toxin.
  • the resulting antisera can be used for the manufacture of a medicament for treating exposure to clostridial neurotoxin.
  • the antisera, or antibodies purified from the antisera can also be used as diagnostic agents to detect either the LHN fragment or the native protein.
  • FIG. 1A shows a 4-12% SDS- PAGE gel in which different fractions are compared for the two cultures. Lanes 1 and 7 are molecular weight markers. Lanes 2-6 contain samples from the BL21 culture, while lanes 8-12 present the corresponding samples from the ER2566 cultures. Lanes 2 and 8 are lysates. The insoluble fractions are shown in lanes 3 and 9, while lanes 4 and 10 are the soluble fractions. The final two lanes in each panel compares whole cells 3 hours after induction (lanes 5 and 11) to whole cells pre-induction (lanes 6 and 12). Figure 1B is the corresponding western blot.
  • strain ER2566 seemed to produce more protein than BL21 by western blot, the results for each strain were similar by ELISA.
  • E. coli BL21 (DE3) (pET26b-LHnA-H - L " ) and E. coli ER2566 (pET26b-LHnA-H " -L ⁇ ) were grown in a Fernbach containing 500 ml YESPG. Cells grew at 37°C to ⁇ OD of 4, then were cooled down to experimental temperature for induction using the auto-induction method. After auto-induction for 3 hours, cells were collected for processing and analyses.
  • Table 2 presents a comparison of the wet culture weights ("WCW”) and OD 6 oo values observed for each strain at induction temperatures of 15, 20, 25, 30, and 35 0 C.
  • Figure 2A shows a 4-12% SDS-PAGE analysis of samples from the E. coli ER2566 (pET 26b-LHnA- L " H) culture. Samples were also western blotted to detect rLH N /A protein. Those results are shown in Figure 2B.
  • lane 1 is the marker.
  • Lane 2 is a pre-induction sample. The odd numbered lanes are soluble fractions, while the even numbered lanes are insoluble fractions for each temperature.
  • Lanes 3 and 4 15°C induction; lanes 5 and 6: 20 0 C induction; lanes 7 and 8: 25 0 C induction; lanes 9 and 10: 3O 0 C induction; and lanes 11 and 12: 35°C induction.
  • the data is presented graphically in Figure 3.
  • the glycerol, antifoam, peptone and yeast extract components are added to 2 liters warm de-ionized water and stirred to dissolve. The volume is adjusted to 3 liters, then the medium is sterilized in autoclave at 122.5 ⁇ 1.5°C for 30 minutes.
  • the phosphate salts are dissolved in 800 mL de-ionized water, and the MgSO 4 is dissolved in 80 mL de-ionized water. Both are then sterilized by autoclaving at 122.5 ⁇ 1.5°C for 20 minutes. After each component has cooled to ambient temperature, 80OmL of the phosphate solution and 8OmL of the MgSO 4 solution are added aseptically to the fermentor.
  • the pH of the medium should be 7.2-7.4, and can be adjusted, if needed, with 40% H 3 PO 4 / 10N NaOH to maintain the appropriate pH.
  • Antibiotic is added via sterile filtration just prior to use. [0100] 2. General Growth and Auto-induction Method
  • the growth conditions were established as follows: temperature to 37°C; agitation started at 200 rpm and set in cascade- controlled mode; dissolved oxygen set at 50% and controlled by agitation; maximal agitation set at 1000 rpm; Airflow set at 0.5 vvm, and pH set at 7.0 and controlled by 40% H 3 PO 4 and 10 N NaOH.
  • the growth temperature was decreased to 16°C or other experimental temperature.
  • agitation was also decreased to 525 rpm to minimize the dissolved oxygen in the growing culture.
  • dissolved oxygen (DO) was maintained at between 10 and 20%, the lowest achievable levels under these conditions.
  • DO dissolved oxygen
  • cells were harvested by centrifugation at 3800 rpm and 4 - 8 0 C in Son/all Centrifuge RC12BP equipped with Sorvall H12000 rotor for 30 minutes. The supernatant solution was disinfected before discard. The cell paste was stored at -70 to -80 °C.
  • FIGS. 6A and 6B Two examples of a fermentation profile are shown Figures 6A and 6B. Each graph shows the OD600, %DO, agitation rate, temperature, and LH N /A (in mg/L) as a function of time. The profiles indicate that the production LH N /A reached maximal levels when the DO started rising again late in the induction period.
  • rl_H N /E For the production of rl_H N /E by recombinant E. coli ER2566 (pET 26b- LHNE (OPT-), seed cultures were grown in two stages before the second seed culture was transferred to a 10-liter Bioflo 310 fermentor. During the initial growth in the fermentor the temperature was kept at 37 0 C. The pH of the culture was maintained at 7.0 by phosphoric acid and sodium hydroxide. The dissolved oxygen (DO) was maintained at 30% by agitation until the agitation rate reached 1200 rpm.
  • DO dissolved oxygen
  • the temperature of the culture was decreased to 16 0 C, airflow was reduced to 0.5 vvm (medium contains 25 g/L glycerol), and agitation was reduced to 500 rpm in order to maintain the DO at the lowest possible level greater than 0.
  • the culture was maintained at 16 0 C, a sparge of 0.5 vvm, and agitation at 500, for 20 more hours.
  • FIGS 8A and 8B show two fermentation profiles for E. coli ER2566 (pET 26b- LH N E (OPT-) in a working volume 10-liter fermentor, Bioflo 310, containing modified Terrific broth and 50 g/L glycerol. The agitation was set at 500 rpm after inoculation and cascade control was used to maintain DO at 30%. The experimental results are summarized in Table 6.
  • E. coli ER2566 pET26b- rl_H N B-H(-)-L(-)
  • induction temperature pET26b- rl_H N B-H(-)-L(-)
  • pET26b- rl_H N B-H(-)-L(-) we also investigated the effect of induction temperature on the soluble rLH N /B protein expression.
  • Cells were grown at 37 0 C in a shake flask containing 2xYT. When the culture reached an A600 of 0.5 to 1.0, IPTG was added and a culture flask was incubated in an incubator shaker i which temperature had been pre-set. Three hours after induction, cells were collected and analyzed by SDS-PAGE and western blot.
  • Figure 9A shows an SDS-PAGE analysis of samples for the production of rl_H N /B by E. coli ER2566 (pET26b- rLH N B-H(-)-L(-) after induction at the indicated temperature for approximately 3-5 hours. Samples were also western blotted to detect ⁇ LHN/B protein. Those results are shown in Figure 9B.
  • lanes 1-6 are soluble fractions of cell lysates in D-PBS+25 mM EDTA after microfluidizer process; lanes 7- 12 are cell paste suspensions in D-PBS+25 mM EDTA.
  • Lanes 1 and 7 15 0 C induction; lanes 2 and 8: 20 0 C induction; lanes 3 and 9: 25°C induction; lanes 4 and 10: 29°C induction; lanes 5 and 11: 33°C induction; and lanes 6 and 12: 37°C induction.
  • E. coli ER2566 pET26b- rl_H N B-H(-)-L(-) obtained from cell bank cryobial culture was inoculated into each of 2 primary seed cultures containing 10 ml_ of Phytone Peptone L-Broth and 30 mg/L kanamycin. After inoculation, these two primary cultures were incubated for 7 hours at 37 °C and 150 rpm. Two milliliter of bulk primary seed was inoculated into each of 5 secondary seed cultures containing 200 mL of Phytone Peptone based production medium, modified Terrific medium.

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Abstract

La présente invention concerne un procédé de production de neurotoxines ou de fragments de neurotoxines de Clostridium solubles, par exemple, de fragments LHN de neurotoxines botuliques. Les protéines produites selon le procédé de l’invention sont utiles, par exemple, en tant qu’agents immunodiagnostiques et en tant que composants vaccinaux.
PCT/US2009/047240 2008-06-12 2009-06-12 Procédé de fermentation pour la production de protéines neurotoxines botuliques solubles WO2009152452A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018200991A1 (fr) * 2017-04-28 2018-11-01 Bonti, Inc. Procédés de production de neurotoxines botuliniques

Citations (4)

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Publication number Priority date Publication date Assignee Title
US4687738A (en) * 1983-03-30 1987-08-18 The Chemo-Sero-Therapeutic Res. Inst. Method for the production of HA fraction containing protective antigens of Bordetella pertussis and pertussis vaccine
WO1998008540A1 (fr) * 1996-08-28 1998-03-05 Ophidian Pharmaceuticals, Inc. VACCIN POLYVALENT CONTRE LA NEUROTOXINE DU $i(CLOSTRIDIUM BOTILINUM)
US20050238668A1 (en) * 2003-09-25 2005-10-27 Allergan, Inc. Media for clostridium bacterium and processes for obtaining a clostridial toxin
WO2006025884A2 (fr) * 2004-05-18 2006-03-09 Biomass Processing Technology, Inc. Fermenteur et procede de fermentation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4687738A (en) * 1983-03-30 1987-08-18 The Chemo-Sero-Therapeutic Res. Inst. Method for the production of HA fraction containing protective antigens of Bordetella pertussis and pertussis vaccine
WO1998008540A1 (fr) * 1996-08-28 1998-03-05 Ophidian Pharmaceuticals, Inc. VACCIN POLYVALENT CONTRE LA NEUROTOXINE DU $i(CLOSTRIDIUM BOTILINUM)
US20050238668A1 (en) * 2003-09-25 2005-10-27 Allergan, Inc. Media for clostridium bacterium and processes for obtaining a clostridial toxin
WO2006025884A2 (fr) * 2004-05-18 2006-03-09 Biomass Processing Technology, Inc. Fermenteur et procede de fermentation

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018200991A1 (fr) * 2017-04-28 2018-11-01 Bonti, Inc. Procédés de production de neurotoxines botuliniques

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